Fu et al. Molecular Cancer (2018) 17:62 https://doi.org/10.1186/s12943-018-0815-z

REVIEW Open Access The critical roles of activated stellate cells- mediated paracrine signaling, metabolism and onco-immunology in pancreatic ductal adenocarcinoma Yaojie Fu1,2, Shanshan Liu1,2, Shan Zeng1,2,3 and Hong Shen1,2,3*

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignant diseases worldwide. It is refractory to conventional treatments, and consequently has a documented 5-year survival rate as low as 7%. Increasing evidence indicates that activated pancreatic stellate cells (PSCs), one of the stromal components in tumor microenvironment (TME), play a crucial part in the desmoplasia, carcinogenesis, aggressiveness, metastasis associated with PDAC. Despite the current understanding of PSCs as a “partner in crime” to PDAC, detailed regulatory roles of PSCs and related microenvironment remain obscure. In addition to multiple paracrine signaling pathways, recent research has confirmed that PSCs-mediated tumor microenvironment may influence behaviors of PDAC via diverse mechanisms, such as rewiring metabolic networks, suppressing immune responses. These new activities are closely linked with treatment and prognosis of PDAC. In this review, we discuss the recent advances regarding new functions of activated PSCs, including PSCs-cancer cells interaction, mechanisms involved in immunosuppressive regulation, and metabolic reprogramming. It’s clear that these updated experimental or clinical studies of PSCs may provide a promising approach for PDAC treatment in the near future. Keywords: Pancreatic stellate cells, PDAC, Metabolic reprogramming, Immune evasion, Drug resistance

Background In recent years, it’s commonly recognized that pancre- Pancreatic ductal adenocarcinoma (PDAC) is an aggres- atic tumor microenvironment (TME) plays a pivotal role sive cancer, which is characterized by rapid progression, in PDAC carcinogenesis, progression and therapeutic re- early metastasis, high recurrence, poor prognosis and sistance [5]. As a key orchestrator of TME, pancreatic stel- limited responsiveness to conventional therapies [1, 2]. late cells (PSCs) aroused considerable attention for its Worldwide, PDAC is becoming increasingly common, and potential value in PDAC therapeutics [6]. In TME, the dy- has a 5-year survival rate of only 7% [3]. Despite numerous namic interactions between cancer cells and PSCs critic- methods in PDAC treatment, including new chemothera- ally manipulate PDAC behaviors via diverse mechanisms. peutic agents, emerging immunotherapy, and advanced sur- The field of PSCs research emerged at the end of 20th gical skills, the long-term survival rate has not shown century. It has been well established that PSCs are respon- significant improvement over the past decade. There are sible for producing the desmoplastic reaction (fibrosis) of few effective therapeutics that can extend the overall sur- PDAC [6–8]. In addition, with exponentially increasing ex- vival of patients with PDAC [4]. perimental data, more details regarding biology and func- tionsofPSCsarecomingtolight[8, 9]. In particular, recent evidence indicated that PSCs exert multiple functions in * Correspondence: [email protected] paracrine actions, metabolic rewiring and intricate immune 1Department of Oncology, Xiangya Hospital, Central South University, Changsha, Hunan 410008, China responses in PDAC. 2Institute of Medical Sciences, Xiangya Hospital, Central South University, Undoubtedly, further explorations on molecular regu- Changsha, Hunan 410008, China latory mechanisms, PSCs-cancer cells interactions, and Full list of author information is available at the end of the article

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fu et al. Molecular Cancer (2018) 17:62 Page 2 of 14

the clinical value of PSCs may benefit patients with PDAC. phenotype [17–19]. Activated PSCs (aPSCs) lose cyto- Targeting PSCs within the TME as a means of inhibiting plastic lipid droplets, and express fibroblast activation PDAC progression is an attractive idea, which may proteins, such as α-smooth muscle actin (α-SMA), and revolutionize PDAC patient treatment and outcome [10]. fibroblast activation protein-α (FAP-α), which serve as biomarkers for aPSCs identification and are negative Phenotypic and functional transition of PSCs prognostic factors in PDAC [17–19]. Meanwhile, aPSCs Pancreatic stellate cells (PSCs), a periacinar star-shaped are the most important cellular source of cancer- stromal cell type in healthy , were successfully associated fibroblasts (CAFs). As a key component in isolated and cultured in 1998 [11, 12]. PSCs share many PDAC stroma, CAFs have high-level heterogeneity, the characteristics with hepatic stellate cells, including morph- distinct subpopulations show complicated effects on ology, storage of lipid droplets rich in vitamin A, locations, growth and progression of PDAC [20, 21]. Moreover, and marker protein expressions [13, 14]. Under homeo- it’s been verified that CAFs derive from diverse cellular static conditions, quiescent pancreatic stellate cells origins, including bone marrow-derived cells (BMDCs), (qPSCs) localize in basolateral aspects of acinar cells, or epithelium, and resident fibroblasts. Actually, CAFs and surround perivascular and periductal regions. qPSCs are aPSCs are different stromal cell populations in PDAC. capable of expressing several protein markers, such as glial Even though both of CAFs and aPSCs share some com- fibrillary acidic protein (GFAP), synemin, and desmin, mon markers, none of these biomarkers are specific most of which are not specific [15, 16]. Even though the [20, 21]. The differences between the CAFs and aPSCs physiological roles of qPSCs haven’t been fully delineated, are still under debate. some functions are postulated and widely recognized. aPSCs also acquire proliferative capacity, and induce These functions include retinoid storage, basic exo-/endo- desmoplastic response via synthesizing abundant extra- crine secretion, maintenance of pancreatic tissue architec- cellular matrix (ECM) [19, 22, 23]. The desmoplastic re- ture, stimulation of amylase secretion, phagocytosis, and action is widely regarded as a hallmark of PDAC, more immunity [16](Table1). In general, qPSCs are believed to importantly, it’s shown to be predominantly responsible contribute to the normal status of the pancreas [15, 16]. for intercellular signaling and TME reprogramming [23] During PDAC, resident qPSCs are activated by some (Fig. 1). However, the contribution of TME-associated risk factors (e.g. ethanol and its metabolites, chronic in- desmoplasia to PDAC growth and progression is still ob- flammation, smoking), environmental stress (e.g. hypo- scure and controversial. The ‘stiff’ stroma impairs the perfusion, hypoxia, oxidative stress), cellular factors (e.g. drug delivery, some investigations indicated that deple- IL-1, IL-6, HIF1α,TGF-β, CCN2) and molecular regulating tion of tumor-associated stroma in mouse PDAC models pathways (e.g. Wnt/β-catenin signaling, PI3K pathway), by using enzymatic degradation of hyaluronic acid (HA) and then transform into an activated myofibroblast-like or Sonic Hedgehog inhibitor IPI926 could suppress

Table 1 Biological comparison of quiescent PSCs (qPSCs) and activated PSCs (aPSCs) Biological behaviors or functions Specific/selective biomarkers qPSCs -Store retinoids in droplets [13, 17] desmin [15, 16] -Function as an immune, progenitor and intermediary cell [16] nestin [15, 16] -Stimulate amylase secretion, phagocytosis and immunity [16] vimentin [18] -Secrete MMPs and TIMPs to maintain ECM turnover; prevent collagens deposition [17] synemin [15, 16] -Produce cytokines, growth factors; basic exo/endocrine secretions in a proper way [18] GFAP [15, 16] -May contribute to acinar regeneration [18] NGF [15, 16] -Involve in maintenance of pancreatic tissue architecture [16, 18] -Help to sustain homeostasis in pancreas microenvironment [16] aPSCs -Induce desmoplastic reactions in TME [19], elevate interstitial pressure [22] α-SMA [17, 18] -Induce hypovascularity and produce excess ECM [19, 23] FAP-α [19] -Contribute to hypoxic and low-nutrient conditions [77, 79] FSP-1 [17–19] -Lose vitamin A lipid vacuoles [18] Fibrinogen [18, 19] -Develop spindle-shaped morphology [17, 18] -Generate growth factors (GFs), cytokines, exosomes, micRNAs that enhance tumor survival, proliferation, migration and metastasis [34, 73, 84, 85] -Promote angiogenesis, PNI and EMT [44, 45, 54, 61] -Mediate chemoresistance and radioresistance [70, 105] -Contribute to complex metabolic networks in TME [112, 115] -Interact with PDA cells or other stromal components [47] -Contribute to immunosuppressive regulations and immune evasion [130–133] Notes: Biological behaviors and functions dramatically change during phenotypic transition of PSCs. Biomarkers of qPSCs are not specific. Abbreviations: aPSCs activated pancreatic stellate cells, qPSCs quiescent pancreatic stellate cells, GFs growth factors, PNI perineural invasion, EMT Epithelial- Mesenchymal Transition, TME tumor microenvironment, GFAP glial fibrillary acidic protein, α-SMA α-smooth muscle actin, NGF nerve growth factor, FAP-α fibroblast activation protein-α, FSP-1 fibroblast-specific protein-1 Fu et al. Molecular Cancer (2018) 17:62 Page 3 of 14

Fig. 1 Phenotypic transition of PSCs and desmoplastic TME. qPSCs are activated by risk factors, local environmental stress, cellular and molecular regulations. During the oncogenesis, aPSCs largely contribute to fibrotic microenvironment, which is a major characteristic of PDAC. The desmoplastic TME consists of epithelial PDA cells and numerous stromal components, such as immunosuppressive cells, aPSCs, collagens and so on

PDAC progression [24, 25]. Oppositely, some new pre- metalloproteinase and its inhibitors (MMPs, TIMPs), cause clinical and clinical data suggested that stromal desmo- sustained fibrosis and create a physical barrier to nutrients plasia acts to restrain, rather than support PDAC or therapies [32, 33]. Recently, more studies suggested that progression [26]. Depletion of myofibroblast and colla- aPSCs play a reciprocal role in the stroma-cancer gen in PDAC displays immunosuppression, enhanced cells interactions, which support PDAC malignant be- tumor hypoxia, EMT program and cancer stem cell-like haviors via inducing drug resistance, metabolic rewiring, phenotype [27]. Activation of Rho-associated protein and immune evasion [33, 34]. kinase2 (ROCK2) signaling can promote PDA cells pro- Collectively, in contrast to qPSCs, aPSCs are morpho- liferation and invasiveness via matrix metalloproteinases logically and functionally transformed. The activated (MMPs) release and collagen degradation [28]. Clinically, phenotype can accelerate TME formation, and frequently high stromal density in resected PDAC was found to be promote PDAC progression through diverse pathways significantly associated with longer disease-free [29]. [35] (Table 1). Taken together, the TME-associated desmoplasia, repre- senting aPSCs activity, plays a dual role in PDAC. Fur- PSCs related diverse paracrine and molecular pathways ther exploration of desmoplastic reaction is really that influence invasion, metastasis, and therapeutic necessary. resistance of PDAC Additionally, persistent PSCs activation results in dra- PSCs are an important source of secretions in TME [7, 36]. matically increased secretion of a wide variety of cyto- A large number of GFs, chemokines, cytokines, miRNAs, kines, chemokines, growth factors (GFs), and exosomes, exosomes and other soluble substances secreted by PSCs, which perform various pathological functions of PDAC. act in an autocrine or paracrine manner to orchestrate con- aPSCs-derived insulin-like growth factor 1 (IGF1), vascu- tinued PSCs activation and signaling transfer between lar endothelial growth factor (VEGF) and platelet-derived stroma and epithelial cancer cells [37–39]. growth factor (PDGF) may promote angiogenesis, epithe- (1) IL-6/JAK/STAT signaling: The presence of chronic lial cancer cells proliferation and migration [16, 30, 31]. inflammation is a hallmark of PDAC progression [40]. The overproduced matrix such as collagens, hyaluronic Recent evidence implicated that PSCs is a main source acid (HA) and unbalanced expression of matrix for large amounts of inflammatory signals. Interleukin- Fu et al. Molecular Cancer (2018) 17:62 Page 4 of 14

6 (IL-6), a cytokine that is produced in abundance by perineural invasion (PNI) [54], and drug resistance [53– components of stroma including PSCs and tumor- 55]. Despite the prevailing notion, that hedgehog-driven associated myeloid cells [41–43], can exert versatile stroma plays a critical role in neoplastic growth and functions to promote PDAC progression. In particular, PDAC progression. Inhibition of SHH signaling seems to IL-6 in TME can activate downstream JAK/STAT sig- enhance delivery of chemotherapy and improve the out- naling via the transmembrane receptor gp130. It’sevi- comes of PDAC [24]. However, some current data pro- denced that IL-6/JAK/STAT signaling axis in TME vided more uncertainties of this opinion, and even shifted plays an important role in the transformation from to the opposite paradigm that SHH signaling may partially pancreatic intraepithelial neoplasia (PanIN) to carcin- act to restrain PDAC growth [26]. The studies demon- oma [44, 45], PDAC aggression, TME remodeling and strated that in spite of the success of IPI926 in treating therapeutic resistance [41, 46]. IL-6/STAT axis activated PDAC mouse models, treatment with SMO inhibitors by aPSCs significantly upregulates some genes expres- alone in PDAC trials showed poor clinical performance sion in PDA cells, such as major EMT regulator Snail, [24]. In contrast, the administration of vascular endothe- mesenchymal marker CDH2, and invasion related genes lial growth factor receptor (VEGFR) blocking antibody se- CCL20, CFB, LCN2 etc, which correlates PDAC migration lectively improved survival of SHH-deficient PDAC, and evolution [42, 47]. Suppressor of cytokine signaling 3 which suggested that SHH signaling-driven stroma may (SOCS3) serves as a potently negative regulator that in- suppress PDAC growth partly by restraining tumor angio- hibits PDA cells migration, invasion, and enhances PDA genesis [26]. Generally speaking, SHH signaling exerts cells apoptosis. Recent study demonstrated that IL-6/ complicated functions in PDAC. More details need to be STAT3 axis could induce SOCS3 methylation via DNMT1, elucidated in the future. which leads to PDAC growth and metastasis [48]. More- (3) Vitamin D Receptor (VDR) pathway: Wide pro- over, PSCs-secreted IL-6 could not only induce PDA cells spective studies have demonstrated that there is a de- proliferation via nuclear factor erythroid 2 (Nrf2)-mediated fined inverse correlation between circulating levels of metabolic reprogramming and reactive oxygen species Vitamin D and risk of developing PDAC or other malig- (ROS) detoxification [48], IL-6/STAT/Nrf2 pathway was nancies [56]. aPSCs express high levels of the VDR. As a also implied to promote EMT in PDAC [47]. notably alternative pathway, Vitamin D Receptor (VDR) Additionally, recent findings suggested the IL-6/JAK/ signaling plays a critical role in driving conversion of STAT3 axis promotes the recruitment of immunosup- qPSCs to their activated state, which then results in stro- pressive cells (e.g. MDSCs, Tregs), which hampers im- mal remodeling of PDAC [57, 58]. Further investigations mune responses of PDAC [49]. shows after treated with Vitamin D analogue Paricalcitol, In general, paracrine IL-6/JAK/STAT signaling plays a PSCs activation can be partly reverted [16, 58]. It reveals pivotal role in PSCs-PDA cells interaction and PDAC that PSCs related VDR pathway may serve as a promis- progression. Pharmacologic blockade of IL-6/JAK/STAT ing molecular target in PDAC therapy [56–58]. signaling may be a new therapeutic strategy for patients (4) CXCL12/CXCR4 signaling axis: PSCs is a pre- with PDAC. dominant source of C-X-C motif chemokine 12 (CXCL12) (2) Paracrine Sonic Hedgehog (SHH) signaling: in TME [59]. High-mobility group box 1 (HMGB1) se- Current study shows paracrine sonic hedgehog (SHH) creted by stressed PDA cells can capture CXCL12 and signaling, which involves both epithelial cancer cells and then form a heterocomplex. It’s evidenced that HMGB1- stromal cells , promotes cancer cells-stroma interactions CXCL12 complex interacts with C-X-C chemokine recep- and ultimately contributes to PDAC progression [50]. tor type 4 (CXCR4), which is highly expressed in PDA To date, it’s clear that paracrine SHH protein, which is cells under hypoxic conditions (or HIF-1α expressed secreted by PDA cells, serves as a hedgehog (HH) path- strongly) [59]. The HMGB1-CXCL12 complex can induce way ligand. SHH signaling is mediated by HH ligand a range of downstream aggressive behaviors, including: (1) binding to the membrane-localized receptor patched gemcitabine treatment resistance [60]; (2) promoting pro- (PTCH) on PSCs, which relieves the inhibitory effect on liferation, EMT, angiogenesis and metastasis of PDA cells a Smoothened (SMO) receptor. Derepressed SMO then [61]; (3) elevating MMP2,9 expression, cancer cells inva- leads to a cascade of cytoplasmic events in PSCs that fa- sion and migration [60–62]; (4) activating other pathways, cilitates the activation of GLI family zinc finger tran- such as PI3K/Akt signaling, Ras/ERK pathway [63]; and scription factors, modulating targeted genes expression (5) blunting immunotherapeutic efficacy, inducing im- and eventually resulting in PSCs activation [51–55]. In munosuppressive status [64]. turn, it’s increasingly apparent that aPSCs regulate TME (5) Other representative paracrine signaling path- remodeling and promote malignant behaviors of PDAC, ways: Apart from mentioned above, various paracrine including driving desmoplastic stroma [52], increased vas- components or intercellular signaling are involved in cularity [26], uncontrolled proliferation [53, 55], PSCs activation and PSCs (or CAFs)-cancer cells Fu et al. Molecular Cancer (2018) 17:62 Page 5 of 14

Table 2 PSCs mainly involved paracrine pathways and their functions Paracrine signaling Mediator(s) Description Functional roles Toll-like receptor (TLR) DAMPs in TME TLR9 is activated both in PDA cells and ·Pro-inflammatory effects [82] signaling PSCs ·Up-regulated expression of PSCs-derived cytokines (e.g. CCL3, CCL11) [83] ·Recruitment of Treg cells in PDAC [83] IL-6/JAK/STAT IL-6 A versatile pathway in PSCs-PDA cells ·Inducing chemoresistance, fibrotic reaction [44, 46] signaling interactions ·Invasive TME remodeling [47] ·Affecting other cytokines production ·Recruitment of immunosuppressive cells [49] ·Enhancing tumor aggressiveness via PSCs-PDA cells crosstalk [44, 47] Shh signaling SHH protein An altered signaling between PSCs ·Sustaining PSCs activation and proliferation [51–55] and tumor cells ·Promoting vasculature and desmoplasia [52] ·Driving perineural invasion (PNI) and drug resistance [53–55] ·Tumor proliferation and progression [51, 53] CXCL12 (SDF-1)/ PSCs-derived SDF-1 It’s highly activated in PDAC, the ·Causing low response to gemcitabine treatment [60] CXCR4 signaling (CXCL12) elevated level is correlated with poor ·Promoting PDAC progression via enhanced clinical outcomes proliferation, EMT, angiogenesis and metastasis [61] ·Inducing over-expressed MMPs, up-regulated invasiveness and migration of tumor cells [60, 62] ·Supporting immunosuppressive environment [64] ·A potential target for PDAC immunotherapy combined with CTLA-4 or PD-L1 checkpoint block [64] MCP-1/CCR2 pathway MCP-1 expressed in PSCs An important cytokine signaling ·Serving as a novel component in PSC inflammatory mediating PSCs activation and and fibrogenic signaling [81] fibrogenic ECM ·Mediating monocytes migration into and then differentiation into PSCs [81] ·Maintaining activated status of PSCs through autocrine manner [15] Ets-2-dependent E26 oncogene homolog 2 New functions unlocked about Est-2 ·Stromal Ets-2 regulates chemokines production and regulation (Ets-2) originated in PSCs signaling in TME of PDAC immune cells recruitment during PDAC ·Fibroblast Ets-2 deletion leads to an increased CD8 +T-cell population, and decreased presence of regulatory T cells (Tregs), MDSCs [74] Peroxisome PPAR-γ ligands A nuclear hormone receptor that is ·Maintenance of quiescent status of PSCs [15, 65] proliferator activated characterized as the master regulator ·PPAR-γ ligand may enhance the phagocytic activity receptor-γ signaling for adiopogenic properties in PSCs of PSCs, which is partially responsible for the (PPAR-γ) inhibition of fibrogenesis [66] Periostin pathway periostin A secretory protein mainly from PSCs, ·Periostin secreted by PSCs creates a tumor-supportive whose expression regulates behaviors microenvironment [67] of both PSCs and TME ·PSCs remains via periostin autocrine loop [68] ·Biphasic effects on PDAC development: low concentration of periostin (to 150 ng/ml) drives mesenchymal-to-epithelial phenotypical transition while high concentration (1μg/ml) promoting cancer cell migration via Akt/PKB signaling pathway [69] microRNAs (miRNAs) Various miRNAs and A recent hot spot, covering many ·Controlling myofibroblast phenotype of PSCs [84] and exosomes exosomes derived from PDA aspects of TME remodeling, PSCs- ·Promoting migration and proliferation of tumor cells cells or PSCs tumor cells interactions [85, 87] ·Mediating metabolic reprogramming, TME remodeling and intracellular interplay [86] ·Delivering nutrients for cancer cells [84] integrin kindlin-2 Newly identified signaling ·Binding of kindlin-2 and integrin, promotes cytokines production in PSCs and further accelerating progression of [39] galectin-1 β-galactoside-binding A heterotrimer protein strongly ·Promoting proliferation and chemokine synthesis of protein expressed in expressed in the stroma of PDAC activated PSCs [70] activated PSCs ·Contributing to the immune escape by enhanced apoptosis and anergy of T cells [71, 72] ·Inducing SDF-1 secreted from PSCs; promoting PDAC metastasis [71, 72] Fu et al. Molecular Cancer (2018) 17:62 Page 6 of 14

Table 2 PSCs mainly involved paracrine pathways and their functions (Continued) Paracrine signaling Mediator(s) Description Functional roles Vitamin D Receptor Circulating Vitamin D A promising target for PDAC ·Mediating PSCs phenotypical switch and stromal (VDR) pathway treatment remodeling [58] ·Enhancing PDAC treatment [58, 108] Growth factors growth factor “Multifunctional messengers” among ·Promoting growth, invasion, migration, and (HGF), Connective tissue all components in TME chemotherapy resistance of PDA cells [34] growth factor (CCN2), others ·Modulator of metabolic crosstalk between tumor cells and stromal components [73] ·PSCs fibrogenic signaling [73] Other signaling HIF-1α, ROS, NF-κB, TGF-β/ Commonly present in numerous ·TME remodeling; promoting proliferation, pathways Smad, VEGF, PDGF, GM-CSF malignancies invasion, migration, chemotherapy resistance, and so on angiogenesis, immune evasion and other behaviors of PDA cells [75–80] Notes: PSCs-related paracrine signaling pathways have been depicted above, including their biological roles, functional molecules and influences on PDAC behaviors interactions (Table 2), including Ca2+ signaling, VEGF, strongly linked to several hallmarks of tumor, such as PDGF, Toll-like receptors (TLRs) signaling, HIF-1α sig- proliferation, metabolism and metastasis [91–93]. In naling, TGF-β/Smad pathways, tumor necrosis factor-α PDAC, tumor interstitial matrix (e.g. collagen, HA) and (TNF-α), monocyte chemoattractant protein-1 (MCP-1), related stromal cells, such as CAFs and PSCs, mainly and periostin, which exert various influences on PDAC contribute to the solid stress. The increased solid stress pathology [15, 34, 65–83] (Table 2). Additionally, the in- is largely responsible for intratumoral vessels compres- volvements of miRNAs and exosomes have recently be- sion, lower perfusion and local hypoxia [24, 94]. More ing reported [84]. For example, PSCs-induced miR-210, importantly, growth-induced solid stress tightly corre- miR-199 upregulation plays an important role in PDA lates with PDAC therapeutic resistance, and strategies cells EMT and migration [85–87], and PSCs-derived designed to alleviate solid stress have the potential to exosomal miR-21 and CCN2 partially drive PSCs fibrotic improve PDAC treatment [94, 95]. It’s implied that fi- signaling [73]. However, further relevant mechanisms brotic and hypovascular stroma reduce drugs delivery still need to be uncovered. via collapsed intratumoral blood vessels, and high intersti- tial fluid pressure (IFP) [96, 97](Fig. 2). Continuous activa- New perspectives on PSCs-mediated molecular mechanisms tion of PSCs (or CAFs) produce various ECM proteins that contribute to metastasis and chemoresistance of PDAC such as HIF-1α, collagen1 (COL1), SHH and PSCs- During dissemination from a primary tumor, TME plays derived periostin, which promote tumor progression, a critical role in determining PDAC invasion and metas- drug irresponsiveness, and contributes to tumor-sup- tasis, regardless of “collective migration” or protease- portive microenvironment and radio-/chemoresistance dependent/independent single tumor cell invasion [88]. [24, 67, 98, 99]. Moreover, a recent study indicated that In particular, the plasticity of PDA cells invasion is fur- the amplified crosstalk among cancer-associated adipo- ther affected by interactions within the tumor stroma, cytes (CAAs), tumor-associated neutrophils (TANs), and where neighboring non-tumor cells contribute to regulating PSCs that occurs in PDAC related obesity leads to an ag- invasion or distant metastasis by a variety of mechanisms. gravation of solid stress, increased tumor progression and Another troublesome problem is therapeutic resistance, reduced chemotherapy response [95]. CAAs generates an which is a major contributor to the poor clinical outcomes inflammatory and fibrotic TME of PDAC. Abundant of PDAC [89]. PSCs can exert multiple functions that are adipocyte-secreted interlukin-1β (IL-1β) mediates obesity- responsible for PDAC invasion, metastasis and drug resist- induced TANs infiltration and PSCs activation in PDAC. ance, such as ECM remodeling, paracrine signaling circuits, Interactions between PSCs and TANs exacerbates desmo- immune regulation, metabolic alterations, local proteolytic plasia in PDAC via angiotensin-II type-1 receptor (AT1) degradation, and angiogenesis [8, 40]. The updated studies signaling, which largely hinders the delivery and efficacy focused on the PSCs’ new contributions to the biological of chemotherapy [95]. behaviors of PDAC are as following (Fig. 2): Apart from the theory of solid stress, another novel (1) ‘Solid stress’ and therapeutic resistance: Elevated mechanism of PSCs-mediated therapeutic resistance has tissue stiffening has become a widely accepted and pas- been recognizing. Transforming growth factor-β (TGF-β) sionately studied biomechanical property of fibrogenic mediates PSCs-expressed cysteine-rich angiogenic in- tumors [90]. The item ‘solid stress’ refers to the physical ducer61 (CYR61), a matricellular protein that negatively forces caused by solid and elastic elements of the extra- regulates nucleoside transporters hENT1 and hCNT3, cellular matrix and cells [90, 91]. Recently, increasing which are responsible for cellular uptake of gemcitabine, evidence suggested that the existence of solid stress is largely reducing chemotherapy responses [100](Fig.2). Fu et al. Molecular Cancer (2018) 17:62 Page 7 of 14

Fig. 2 PSCs mediate invasion, metastasis, therapeutic resistance of PDAC. Multiple factors are involved in, such as immune evasion, metabolic reprogramming, ECM remodeling, various paracrine signaling and so forth

(2) Newly identified PSCs-expressed miRNAs and PI3K/AKT/mTOR signaling pathway [103], which may PDAC progression: miRNAs have become a hot spot be explored to treat PSC-related pathologies and to de- for cancer research [101]. In PDAC, some new investiga- velop anti-fibrotic approaches. Another therapeutic agent, tions highlighted the roles of PSC-expressing miRNAs in Tocotrienols, selectively trigger aPSCs apoptosis and au- controlling the myofibroblast phenotype of PSCs and tophagic death by targeting the mitochondrial permeabil- their influences on migration and proliferation of tumor ity transition pore [104]. It also unveils another potential cells. For example, miR-210 was reported to regulate the approach to ameliorate the fibrogenesis in PDAC. interactions between PSCs and PDA cells via ERK and Akt pathways [85]. Moreover, recent studies validated that miR-199a and miR-214 are upregulated in patient-derived aPSCs in PDAC metabolic reprogramming pancreatic PSCs, and targeting them caused the dediffer- The TME of PDAC is the major source of both interstitial entiation of aPSCs and inhibited tumor-promoting para- pressure and oxidative stress [105]. High concentration of crine effects [86]. the PSCs-derived matrix including hyaluronic acid (HA), (3) Autophagy in aPSCs: A potential target for collagen and glycosaminoglycan, contributes to the dense PDAC: Besides revealed involvement of PSCs in tumor fibrotic stroma and subsequently leads to intense pressure invasion and metastasis (e.g. MMPs activities, EMT, in TME [106]. As a result, elevated stromal pressure angiogenesis, signaling pathways and so on), new in vivo causes vascular collapse, tumor tissue hypo-perfusion, and findings suggested that autophagy in PSCs, which is in- a lack of oxygen and nutrient delivery [105, 107, 108]. The duced by environmental stress and PDA cells-stroma in- environmental stress imposes “energy crisis” to cancer teractions, is strongly associated with tumor T category cells. Despite diverse mechanisms promoting extracellular histologic grade, peritoneal dissemination, perivascular glucose acquisition via the Warburg or the reverse invasion and lymph node metastasis [102]. This novel Warburg effect in cancer cells (e.g. HIF-1α signaling, discovery might be a goal of therapeutic interest, and over-expression of aerobic glycolytic enzymes like NF-kB, predict the hypothesis that targeting autophagy could be MCTs, PKM1/2) [108–111], obviously, enhanced glucose a promising candidate for treatment strategies in PDAC. metabolism cannot compensate for energetic and biosyn- Coenzyme Q10 (CoQ10), commonly known as ubiquin- thetic shortfall completely. To sustain macromolecular one, has been suggested to inhibit the activation of biosynthesis and better tumor survival, metabolic rewiring aPSCs by suppressing the autophagy through activating among cancer cells and stromal components enables Fu et al. Molecular Cancer (2018) 17:62 Page 8 of 14

access to recycling nutritional substrates and alternative cells proliferation [119]. It’s demonstrated that onco- fuel sources for growth [112, 113]. More importantly, genic KRAS signaling mainly drives Gln conversion into accumulating studies suggest that PSCs can strikingly re- aspartate for further energy generation and anabolism by program metabolic machinery for PDAC, especially the activating the GOT2/GOT1/ME1 pathway, while at the metabolic crosstalk between PSCs and PDA cells, therefore same time, initiating glutathione (GSH) and NADPH facilitating PDAC progression and invasiveness under biosynthesis, and inhibiting Nrf2/ME1/ROS activities to nutrient-limiting conditions [114, 115]. In general, under- sustain cellular redox balance and enhance cytoprotec- standing more details about metabolic reciprocation be- tion of cancer cells [113, 115, 120]. tween epithelial cancer cells and aPSCs seems to be crucial. Recently, it’s become increasingly apparent that the It’s recognized that besides environmental stresses, meta- tumor cell KRAS mutation manipulates signaling within bolic interplay between PDA cells and PSCs is the conse- both PDA cells and adjacent PSCs, and influences PSCs- quence of genetic mutations combined with comprehensive tumoral metabolic interactions. KRAS rapidly promotes paracrine signaling regulations [108, 115, 116](Fig.3). sonic hedgehog (SHH) secretion from PDA cells, which Multiple genes (e.g. TP53, Myc, Smad4, CDKN2A) can activates PSCs to induce widespread events such as over- drive PDAC metabolic reprogramming to meet the de- expression of IGF1, GAS6, GM-CSF and other cytokines. mands of tumor-relevant anabolic processes under ster- This results in PSCs reciprocally sending a feedback sig- ile conditions [108, 117]. Among these, oncogenic KRAS naling to PDA cells via IGF1R/AXL axis, activating mutation signaling has been recently shown to play a downstream PI3K-Akt phosphorylation and increasing predominant role in multiple aspects of PDAC metabol- spare mitochondrial respiratory capacity in PDA cells ism, including adaptive metabolic responses and cancer [121], which elevates oxygen availability for PDA cells cells-PSCs mutualism [115, 118]. More evidence has under hypoxia. Additionally, KRAS-mutant PDA cells emerged that the KRAS mutation not only enhances glu- upregulate macropinocytosis, an endocytosis-mediated cose uptake, but also activates expression of several key bulk uptake, to import extracellular proteins, which is enzymes in aerobic glycolysis (the Warburg effect). Fur- ultimately delivered to lysosomes for catabolism, fueling thermore, KRAS-driven glutamine (Gln) metabolism be- TCA cycle, essential amino acids recycling and support- comes a major source of carbon and nitrogen for cancer ing cell growth [122–124].

Fig. 3 PSCs in metabolic reprogramming. In KRAS-dependent pathways, diverse cytokines and signaling pathways mediate metabolic interactions between PSCs and PDA cells. KRAS-driven glutamine (Gln) metabolism becomes a major carbon source for tumor cells survival; PSCs-derived IGF elevates mitochondrial respiration in PDA cells via IGF1R/AXL axis; KRAS-mutant PDA cells can obtain extracellular proteins for supporting growth through upregulated macropinocytosis. PSCs-secreted non-essential amino acids (NEAAs), such as autophagy-induced Ala, can serve as an alternative energy source to fuel PDA cells. In KRAS-independent pathways, PSCs-derived growth factors (GFs) and exosomes play a pivotal role in mediating survival, proliferation, metastasis, biosynthesis of tumor cells Fu et al. Molecular Cancer (2018) 17:62 Page 9 of 14

More interestingly, PSCs-derived non-essential amino acids (NEAAs) also provide nutrients to feed PDA cells. Recent studies revealed that PDA cells increase autophagy in PSCs via unclear mechanisms [125], and then mediate PSCs secreting alanine (Ala) [125, 126]. As a linkage of this cooperative metabolic relationship, PSCs-derived Ala is taken up by PDA cells and acts as an alterna- tive carbon source to glucose/glutamine, shunts glu- cose to Ser/Gly biosynthesis, and supports lipid and NEAAs biosynthesis [115, 125]. In contrast to KRAS signaling related rewiring men- tioned above, metabolic reprograming via KRAS-inde- pendent pathways has been identified [126]. GFs (e.g. connective tissue growth factor; CTGF and fibroblast growth factor-2; FGF2) and cytokines exchange between cancer cells and surrounding PSCs have been proved to be pivotal in metabolic crosstalk [126–128]. Furthermore, PSCs-derived exosomes contain various biomolecules, in- cluding mRNA, miRNA, intracellular metabolites (e.g. amino acids, acetate, stearate, palmitate, and lactate), Fig. 4 Immunosuppressive modulator role of PSCs. PSCs induce which enter PDA cells, both fuel the tricarboxylic acid TME remodeling, dense matrix caused hypoxia and hypo-vascularity impair T cells infiltration and their nutrition obtaining; multifactorial cycle (TCA cycle) and enhance tumor growth in a manner T cell exhaustion attenuates Teff functions; PSCs-derived suppressive similar to macropinocytosis [84, 126, 128, 129]. factors (such as IL-6, CXCL12), suppressive signaling and recruitment

Collectively, the description of these new metabolic of suppressive cells (such as MDSCs, Treg cells, TAMs) create an crosstalk pathways further highlights that (1) PSCs play immunosuppressive TME in PDAC a key role in intra-tumoral metabolic networks, and (2) the PDA cells-PSCs metabolic “coupling” contributes significantly to PDAC development under nutrient-poor antigen sensitivity, cytokine production and migration environment [115]. [138]. FAK1 level is elevated in PDA cells and correlates with robust fibrosis and poor CD8+ T cell accumulation. PSCs-mediated immunosuppressive microenvironment in The rigid ECM components secreted by PSCs, such as anticancer immunity collagen or fibronectin, induce Rho-associated coiled-coil Despite continuous progress in understanding the kinase-dependent activation of FAK1, greatly contributing immune-dependent regulations of PDAC and the devel- to suppressed anticancer immunity [138]. opment of immunotherapies [130], therapeutic advances Second, desmoplastic response creates hypoxic and have been insufficient [131, 132]. Elucidating a method avascular conditions, which imposes considerable ener- to enhance antitumor immunity and immunotherapy getic constraints on tumor cells, PSCs and immune cells seems to be a challenge in PDAC treatment. Immune [115]. As we mentioned above, PSCs constitute the major evasion and T cell dysfunction can be mediated by a var- source of cancer-associated fibroblasts (CAFs) in PDAC. iety of mechanisms, such as the immunosuppressive Paracrine signaling from neighbor PSCs (or CAFs) and microenvironment in PDAC patients, which involves in- CAFs-tumor cells interactions lead to metabolic repro- teractions among tumor cells, infiltrated immune cells gramming, by which cancer cells express more nutrients and stromal components [108, 117], and makes a major import molecules (e.g. GLUT1, MCTs, ASCT2, LATs) hurdle for immune responses [133]. As a currently com- to obtain fuel sources for survival [139]. Elevated pelling role, PSCs display multiple effects on immuno- indoleamine-2,3 dioxygenase 1 (IDO1) and arginase suppressive regulation that makes PDAC therapeutics (ARG1, ARG2) in metabolically altered CAFs may deplete more difficult [134, 135] (Fig. 4). tryptophan and arginine, which are crucial for T effector + + First, α-SMA or FAP PSCs play a pivotal role in (Teff)cells’ proliferation and activation [139, 140]. Mean- TME remodeling. PSCs-mediated desmoplasia results in while, confronted with “metabolic competition”,lackof excess matrix deposition in TME, which has been postu- glucose impairs T cell’s anti-tumor immunity and secre- lated to limit T cell infiltration and function once recruited tion of Interferon-γ (IFN-γ)[141, 142], while low lipid into tumor site [136, 137]. A striking cell-intrinsic path- support results in TNF receptor associated factor 6 way impacting cancer immunity is focal adhesion kinase (TRAF6) deficiency, which inhibits long-lasting memory (FAK), a tyrosine kinase that regulates T cell survival, CD8+ T cells formation [143]. Fu et al. Molecular Cancer (2018) 17:62 Page 10 of 14

Significantly, it has been commonly assumed that T recognized as a main driver for PDAC progression. Al- cells in the context of established progressing cancer pa- though development on basic studies and therapeutic tients exhibit an exhausted status (termed as “T cell ex- strategies targeting PSCs have been revealing, more haustion”) due to various factors, such as persistent details on PSCs and PDAC treatment remains to be tumor antigen load, inhibitory checkpoint signaling illustrated. It’s promising that further understanding pathways (e.g. PD-1, LAG-3, CTLA-4), cell-intrinsic tol- about PSCs will open new avenues for translational erance programs, and, more importantly, the complex medicine and more meaningful clinical therapies for immunosuppressive environment [144]. In PDAC, PSCs PDAC. to a large extent mediate physiological changes in TME (e.g. hypoxia and low pH). HIF-1α activation can sup- Abbreviations Ala: Alanine; aPSCs: activated pancreatic stellate cells; ARG: Arginase; press immunity or promote tumor evasion, however the AT1: Angiotensin-II type-1 receptor; BMDCs: Bone marrow-derived cells; underlying molecular mechanisms remain to be further CAAs: Cancer-associated adipocytes; CAFs: Cancer-associated fibroblasts; identified [145, 146]. COL1: Collagen 1; CoQ10: Coenzyme Q10; CTGF: Connective tissue growth factor; CTL: Cytotoxic T lymphocyte; CTLA-4: Cytotoxic T lymphocyte- PSCs also secrete numerous soluble cytokines that associated antigen-4; CXCL12: C-X-C motif chemokine 12; CXCR4: C-X-C contribute to “T cell exhaustion” and dysfunction. It’s chemokine receptor type 4; CYR61: Cysteine-rich angiogenic inducer61; well evidenced that PSCs-derived CXCL12 (also named ECM: Extracellular matrix; ERK: Extracellular signal-regulated kinase; FAK: Focal adhesion kinase; FAP-α: Fibroblast activation protein-α; FGF2: Fibroblast stromal-derived factor-1, SDF-1) can limit cytotoxic T growth factor-2; FSP-1: Fibroblast-specific protein-1; GFAP: Glial fibrillary cells trafficking, mediate macrophages’ differentiation acidic protein; GFs: Growth factors; Gln: Glutamine; Gly: Glycine; into a pro-tumor M2 phenotype (tumor-promiting), and GOT1: Aspartate aminotransferase 1; GOT2: Aspartate aminotransferase 2; GSH: Glutathione; HA: Hyaluronic acid; HH: Hedgehog; HIF-1α: Hypoxia recruit myeloid-derived suppressor cells (MDSCs), inducible factor 1α; HMGB1: High-mobility group box 1; IDO1: Indoleamine- tumor-associated neutrophils to the tumor site [147]. At 2,3 dioxygenase 1; IFN-γ: Interferon-γ; IFP: Interstitial fluid pressure; the same time, CXCL12/ SDF-1 bound to PDA cells can IGF1: Insulin-like growth factor 1; IL-1: Interleukin-1; IL10: Interleukin-10; IL- 6: Interleukin-6; JAK: Janus kinases; LAG-3: Lymphocyte-activation gene 3; inhibit T cell access to immune responses [148]. Recent MCP-1: Monocyte chemoattractant protein-1; MCTs: Monocarboxylate clinical trials demonstrated that inhibiting CXCR4, a transporters; MDSCs: Myeloid-derived suppressor cells; ME1: Malic enzyme 1; CXCL12 receptor, can dramatically promote T-cell accu- miRNAs: microRNAs; MMPs: Matrix metalloproteinases; NADPH: Hicotinamide adenine dinucleotide phosphate; NEAAs: Non-essential amino acids; NF- mulation and synergize with the checkpoint antagonist, kB: Nuclear factor-kB; Nrf2: Nuclear factor erythroid 2; PD-1: Programmed cell α-PD-L1, to cause cancer regression [64, 148]. death protein 1; PDAC: Pancreatic ductal adenocarcinoma; PDGF: Platelet- Similarly, another versatile PSCs/MDSCs derived pro- derived growth factor; PI3K: Phosphatidylinositol 3 kinase; PKM1/2: Pyruvate kinase M 1 and 2; PNI: Perineural invasion; PSCs: Pancreatic stellate cells; inflammatory cytokine, interleukine-6 (IL-6), can sup- PanIN: Pancreatic intraepithelial neoplasia; qPSCs: quiescent pancreatic press cytotoxic T lymphocyte (CTL)-driven antitumor stellate cells; ROCK2: Rho-associated protein kinase2; ROS: Reactive oxygen immunity by multiple mechanisms, including impairing species; SDF-1: Stromal-derived factor-1; Ser: Serine; SHH: Sonic hedgehog; SMO: Smoothened; SOCS3: Suppressor of cytokine signaling 3; STAT: Signal Teff cells trans-endothelial migration, activation of Treg transducer and activator of transcription; TANs: Tumor-associated neutrophils; cells Foxp3+ or tumor-associated macrophages (TAMs), Teff: T effector cells; TGF-β: Transforming growth factor-β; TIMPs: Tissue inhibitors of metalloproteinases; TLRs: Toll-like receptors; TME: Tumor and inducing imbalance of Treg/Teff activities [149, 150]. microenvironment; TNF-α: Tumor necrosis factor-α; TRAF6: TNF receptor Moreover, large amounts of PSCs-derived suppressive associated factor 6; Treg: Regulatory T cell; VDR: Vitamin D Receptor; cytokines such as IL10, TGF-β, VEGF, MCP-1, GM-CSF, VEGF: Vascular endothelial growth factor; VEGFR: Vascular endothelial growth factor receptor; α-SMA: PGE2, also contribute largely to immune evasion and α-smooth muscle actin anti-tumor hyporesponsiveness of PDAC [144, 151].

In short, with regard to the immunity regulation in Acknowledgements PDAC, PSCs seem to be a powerful immunosuppressive The authors thank Dr. Xingyu Li for assisting in drafting the tables. modulator via numerous pathways. Targeting PSCs may pave a novel avenue for enhancing immunotherapies for Funding This study was supported by grants from the National Natural Science PDAC. Foundation of China (No. 81172470, 81070362, 81372629 & 81772627), and two key projects from the Nature Science Foundation of Hunan Province Conclusions (No. 2015JC3021 & No. 2016JC2037). PSCs surrounding tumor cells is an emerging stromal component that has been receiving huge attention re- Availability of data and materials Not applicable. cently. As a powerful tumor contributor, there is accu- mulating evidence supporting the multiple roles of PSCs Authors’ contributions in the establishment of TME, such as regulating envir- YF, SL, SZ and HS designed and drafted the manuscript; YF, SZ and HS wrote onmental homeostasis and metabolic reprogramming, figure legends and revised the article; SL drew the figures. All authors read and approved the final manuscript. supporting tumor survival, immune evasion, invasion, distant metastasis and therapeutic resistance. The inter- Ethics approval and consent to participate play between cancer cells and PSCs is increasingly Not applicable. Fu et al. Molecular Cancer (2018) 17:62 Page 11 of 14

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